title: Characterization of a multifunctional PEG-based gene delivery system containing nuclear localization signals and endosomal escape peptides.
abstract: Endosomal escape and nuclear localization are two barriers to gene delivery that need to be addressed in the design of new nonviral gene delivery vehicles. We have previously synthesized low-toxicity polyethylene glycol (PEG)-based vehicles with endosomal escape functionalities, but it was determined that the transfection efficiency of PEG-based vehicles that escaped the endosome was still limited by poor nuclear localization. Two different nuclear localization signal (NLS) peptides, SV40 and TAT, were coupled to PEG-based vehicles with DNA-binding peptides (DBPs) to determine the effect of NLS peptides on the transfection efficiency of PEG-based gene delivery vehicles. Coupling one SV40 peptide, a classical NLS, or two TAT peptides, a nonclassical NLS, to PEG-DBP vehicles increased the transfection efficiency of PEG-DBP/DNA particles 15-fold and resulted in similar efficiency to that of a common cationic polymer vehicle, polyethylenimine (PEI). The transfection efficiency of both types of PEG-DBP-NLS particles was further increased 7-fold in the presence of chloroquine, suggesting that the transfection efficiency of PEG-DBP-NLS particles is limited by their ability to escape the endosome. To develop particles that could escape the endosome and target the nucleus, a mixture of PEG-DBP-NLS vehicles and PEG-based vehicles with DBPs and endosomal escape peptides were complexed with plasmid DNA to form multifunctional particles that had a transfection efficiency 2-3 times higher than that of PEI. Additionally, the PEG-based vehicles were less toxic and more resistant to nonspecific protein adsorption than PEI, making them an attractive alternative for nonviral gene delivery.
authors.affiliation: Department of Environmental, Energy, and Chemical Engineering, Washington University, One Brookings Drive, Saint Louis, MO 63130-4899, USA.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Moore
authors.forename: Clayton L
authors.initials: CL
authors.lastname: Sheppard
authors.forename: Shelly E
authors.initials: SE
authors.lastname: Sakiyama-Elbert
mesh_terms: D000818:Animals; D002021:Buffers; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D011992:Endosomes / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D019913:Nuclear Localization Signals / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D010710:Phosphates / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D011094:Polyethyleneimine / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D002021:Buffers; C526981:INF7 peptide; D019913:Nuclear Localization Signals; D010455:Peptides; D010710:Phosphates; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D011094:Polyethyleneimine